GSM6809891: KAT5, multi, ncATAC-seq; Mus musculus; ATAC-seq
Frozen tissue was homogenized in 100 µL homogenization buffer NP+ (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris⋅HCl, pH 7.4; plus 1 mM DTT, 1X protease inhibitor 50X, 1 U/uL RiboLock RNAse inhibitor, 0.1% NP-40, in H20), filtered through a 40 µm cell strainer and washed with 500 µL homogenization buffer (no NP-40). Samples were centrifuged at 1000g for 8 min at 4 °C. The pellet was resuspended in 210 µL homogenization buffer (no NP-40), and 290 µL of 50% Iodixanol solution (Iodixanol 60%, IDM 1X, 1 mM DTT, 1X protease inhibitor 50X, 1 U/uL RiboLock RNAse inhibitor) was added. Nuclei suspension was transferred to a tube (ref:344090, Beckman), completed with a layer of 100 µL homogenization buffer (no NP-40) and centrifuged at 10000g for 22 min at 4 °C using SW 55 Ti rotor, Optima XL-80K ultracentrifuge. Pellet was resuspended in 100 µL of permeabilization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, pH 7.4; plus 1 mM DTT, 1X protease inhibitor 50X, 1 U/uL RiboLock RNAse inhibitor, 0,01%  Tween, 0,001% Digitonin, 0,01% NP-40, 0,5% BSA, in H20)  for 2 min at 4 °C, 500 µL of wash buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris⋅HCl, pH 7.4; plus 1 mM DTT, 1X protease inhibitor 50X, 1 U/uL RiboLock RNAse inhibitor, 0,5% BSA, in H20) were added and samples were centrifuged at 1000g for 10 min at 4 °C. Pellets were resuspended in diluted nuclei buffer (see Chromium nuclei isolation kit, 10X Genomics) and filtered through a 40 µm Flowmi strainer. Final sample concentrations were eGFP: 4040 nuclei/uL, CBP: 3645 nuclei/uL, KAT5: 2925 nuclei/uL. Samples were immediately processed for Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Chromium Next GEM Single Cell Multiome ATAC + Gene Expression